The three related trypanosomatid pathogens, Trypanosoma brucei spec., Trypanosoma cruzi, and Leishmania spec., are the causative agents of African sleeping sickness, Chagas1 disease, and Leishmaniasis, respectively. These important pathogens affect large populations globally and cause numerous deaths. The drugs for these diseases are unsuitable since they are very toxic, not very effective, and resistance to these drugs has developed. Mitochondrial gene expression in trypanosomatids requires a particular form of RNA editing, unique to these organisms, by which precursor mRNA sequences are changed, often extensively, by the insertion and the deletion of uridine nucleotides. The edited mRNAs are translated into components of the oxidative phosphorylation system. Our finding that mitochondrial RNA editing is essential in the disease-causing stage of T. brucei has suggested a new promising target for anti- trypanosomatid drugs. Editing is catalyzed by a multiprotein complex (editosome) that has not yet been fully defined or characterized. This project proposes to generate chemical compounds to characterize the editosome proteins and determine their potential as targets for drugs that will be effective against the three related trypanosomatid pathogens. We are planning to use our expertise in the RNA biochemistry and adapt our ribozyme based assay for high throughput screening of compounds against the editosome. Specific Aim 1 outlines the plan to develop a high throughput screen using fluorescence resonance energy transfer (FRET) to report on the essential functions of the editosome. Specific Aim 2 will describe the secondary assays to validate the specificity of inhibition observed in the first aim. These studies will potentially identify compounds that may selectively perturb editosome assembly and/or function and characterize the various interactions and reaction stages of the RNA editing. They will also contribute to development of drugs against trypanosomatids.